P. aeruginosa is one of the most virulent opportunistic pathogens of man. The morbidity of P. aeruginosa infections results from the ability of the bacterium to colonize previously injured or disrupted epithelial cell layers, cause further epithelial cell damage, and in many cases, gain access to other tissues or the blood stream. To dissect these steps, the investigators make use of a recently developed in vitro and a recently developed animal model of acute pneumonia in which localized cytotoxicity in a polarized epithelial cell line grown in vitro correlates with virulence in this animal model of acute pneumonia. These events do not appear to depend on previously identified virulence factors with the possible exception of exoenzyme S. In preliminary work, they carried out a genetic screen of transposon mutants of a cytotoxic and animal-virulent human isolate of P. aeruginosa (PA103) to identify genes that mediate this localized cytotoxicity. In this grant, the investigators will explore the role revealed by each of these mutants in localized cytotoxicity in an in vitro epithelial cell model of P. aeruginosa-mediated epithelial cell damage and will test them in an animal model of acute pneumonia. Specifically, (i) They will investigate the site of action, function and structure/function relationship, regulation, cellular substrates, and virulence in an animal model of acute pneumonia of the putative novel cytotoxin (PepA) they have discovered. (ii) They hypothesize that either invasion is necessary for and precedes cytotoxicity or that these two events are mutually exclusive. They will test these hypotheses by genetic and pharmacological approaches. (iii) Their initial results demonstrate that while functional pili are necessary for P. aeruginosa-mediated epithelial cell damage in vitro, this function is independent of their ability to bind to the apical surface of MDCK cells. They propose experiments to test further confirm these results. (iv) They postulate that the role of pili in function in cytotoxicity is to allow bacteria to aggregate before binding the apical surface, to move along the surface of epithelial cells by twitching motility, or to transduce the signal that initiates contact-mediated secretion of the putative effectors of cytotoxicity by type III secretion. To test these hypotheses, they will employ time-lapse videomicroscopy to examine the ability of the bacteria to aggregate prior to mediating cell injury, they will use genetic approaches to determine whether twitching motility can be separated from cytotoxicity, and they will test whether pili function to transmit the signal that host cell binding has occurred, leading to contact-mediated type III secretion of PepA. From these studies will come novel targets for anti-Pseudomonal therapies and a better understanding how this bacterium colonizes and injures epithelial cells. In addition, the study of the complex interplay between P. aeruginosa and the host epithelium may reveal new insights into epithelial cell biology.